High-throughput mapping of antigen specificity to B-cell-receptor sequence for characterizing antibody responses in HIV-vaccinated and infected individuals

项目摘要。寻找有效的HIV-1疫苗仍然是当务之急，而且还有更深层次的 了解免疫系统如何识别HIV-1有助于指导疫苗设计。最近，很多 努力的重点是了解抗体对艾滋病毒-1感染和疫苗接种的反应，因为抗体 已被证明在治疗和预防中有用，并作为抗体特异性疫苗设计的模板。而当 HIV-1抗体反应是多克隆和复杂的，下一代测序(NGS)的进展 技术使我们能够以前所未有的分辨率看到这种多克隆反应，作为 单个单抗序列。序列鉴定通常之后是功能性抗体 鉴定，其中一个主要组成部分是抗原/表位特异性的映射。 标准抗体分析流水线的一个主要挑战是序列识别和 抗体的功能表征过程通常是分离的。这防止了真正的高- 抗体-抗原特异性的吞吐量图谱，仅为一小部分 从任何给定样本中选择的抗体序列。为了应对这一挑战，我们在这里建议开发一种 一种技术，对于给定的样本，将能够将抗体序列映射到来自 单次高通量实验。Libra-seq(将B细胞受体与抗原特异性联系起来)技术 通过测序)，包括物理混合B细胞样本和(理论上没有限制的)池 条形码抗原，从而能够同时恢复：(I)配对的重链-轻链BCR序列 和(Ii)特定B细胞的抗原特异性。特别是，这一技术开发项目将广泛关注 在两个特定目标上：在特定目标1中，我们将评估不同的抗原条形码策略和 其他分析变量对天秤座的准确性和性能的影响。这个目标的目标是优化天秤座- 从测序实验中准确检测bcr序列和抗原特异性的能力。在……里面 具体目标2，我们的目标是同时映射给定的HIV特异性B细胞的目标表位，通过 筛选具有表位敲除突变的抗原和野生型抗原的鸡尾酒。这些 这些努力不仅将导致识别艾滋病毒特异性B细胞，而且还将提供残留量 关于同一高通量实验中抗原的特定表位靶标的信息。 最终，对于给定的感染或疫苗样本，Libra-seq技术将提供 为了恢复数万到数十万个B细胞的抗体序列和抗原特异性 细胞水平。为了证明Libra-seq的实用性，我们将对HIV-1感染和 疫苗接种队列。更广泛地说，Libra-seq将是高效和准确的B细胞的不可或缺的工具 分析，有可能对疫苗和抗体发现领域产生广泛影响，不仅是对艾滋病毒-1 而且对其他具有生物医学意义的病原体也有广泛的影响。

Project Summary. The search for an effective HIV-1 vaccine remains a top priority, and a deeper understanding of how the immune system recognizes HIV-1 can help inform vaccine design. Lately, much effort has focused on understanding antibody responses to HIV-1 infection and vaccination, since antibodies have proven useful in therapy and prevention, and as templates for antibody-specific vaccine design. While antibody responses to HIV-1 are polyclonal and complex, advances in next-generation sequencing (NGS) technologies enable us to see such polyclonal responses at an unprecedented resolution, as a collection of individual monoclonal antibody sequences. Sequence identification is typically followed by functional antibody characterization, a primary component of which is the mapping of antigen/epitope specificity. A major challenge with the standard antibody analysis pipeline is that the sequence identification and functional characterization processes for antibodies are generally decoupled. This prevents truly high- throughput mapping of antibody-antigen specificity, providing only limited information for a small subset of selected antibody sequences from any given sample. To address this challenge, here we propose to develop a technology that, for a given sample, will enable the mapping of antibody sequence to antigen specificity from a single high-throughput experiment. The technology, LIBRA-seq (LInking B-cell Receptor to Antigen specificity through sequencing), involves physically mixing a B-cell sample with a (theoretically unlimited) pool of barcoded antigens, thus enabling the simultaneous recovery of: (i) paired heavy-light chain BCR sequences and (ii) antigen specificity for a given B cell. In particular, this technology development project will broadly focus on two specific aims: In Specific Aim 1, we will evaluate the effect of different antigen barcoding strategies and other assay variables on LIBRA-seq accuracy and performance. The goal in this aim is to optimize the LIBRA- seq ability to accurately detect BCR sequence and antigen specificity from a sequencing experiment. In Specific Aim 2, we will aim to simultaneously map the target epitope of a given HIV-specific B cell, by screening a cocktail of antigens with epitope-knockout mutations along with the wildtype antigens. These efforts will not only lead to the identification of HIV-specific B cells, but will also provide residue-level information about the specific epitope target on the antigen from the same high-throughput experiment. Ultimately, for a given infection or vaccination sample, the LIBRA-seq technology will provide the ability to recover antibody sequence and antigen specificity for tens to hundreds of thousands of B cells at the single- cell level. To demonstrate the utility of LIBRA-seq, we will characterize samples from HIV-1 infection and vaccination cohorts. More generally, LIBRA-seq will be an integral tool for efficient and accurate B-cell analysis, with the potential for broad impact on the fields of vaccine and antibody discovery not only for HIV-1 but also for a wide range of other pathogens of biomedical significance.